Molecular motor proteins operate in a complex and crowded cellular environment. Microtubules in the cell are coated with microtubule-associated proteins (MAPs) and serve as tracks for the simultaneous movement of many types of organelles and vesicles. In addition, many critical cellular processes such as endocytosis involve the regulated switching of transport organelles from one type of cytoskeletal filament to another. During endocytosis, vesicles make a directed switch from movement along cortical actin filaments to centripetal movement along microtubules toward the cell center. Initial in vitro systems to analyze motor function have involved the study of single motor types moving along naked filaments polymerized from purified actin or tubulin subunits. However, in vitro studies using increasingly more complex models will provide insights that more clearly address the cellular situation. Here, we propose to build on recent data demonstrating that motors are differentially affected by obstacles in their paths, such as MAPs or intersecting filaments. We will compare motor function at the single molecule level and also in the context of endogenous cellular cargos such as purified endocytic vesicles. Specifically, we propose the following three aims: (1) To investigate the effects of microtubule associated proteins (MAPs) on the motility of the microtubule motors kinesin and dynein functioning either individually or in concert on purified vesicles and organelles. Can microtubule-associated proteins provide spatially-specific regulation of motor function in the cell? (2) To examine the interactions of multiple motor proteins, including both actin-based and microtubule-based motors bound to cargos moving on arrays composed of intersecting actin filaments and microtubules, to examine the parameters associated with track switching. Is filament switching a regulated or a stochastic process in the cell? And (3) To compare these in vitro observations with cellular studies examining motor function during receptor-mediated endocytosis with three-dimensional, high resolution fluorescence imaging coupled to EM analysis to obtain spatial and temporal information on filament switching in the cell. Together, these studies will provide new insights into motor integration and coordination during intracelllular transport.